Synthesis and evaluation of novel bifunctional chelating agents based on 1,4,7,10-Tetraazacyclododecane-N,N,N,N-Tetraacetic acid for radiolabeling proteins L.L. Chappell, D. Ma, D.E. Milenic, K. Garmestani, V. Venditto, M.P. Beitzel, M.W. Brechbiel* Radioimmune & Inorganic Chemistry Section, ROB, CCR, NCI, NIH, Bethesda, MD, 20892-1002, USA Received 1 November 2002; received in revised form 27 January 2003; accepted 15 February 2003 Abstract Detailed synthesis of the bifunctional chelating agents 2-methyl-6-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (1B4M-DOTA) and 2-(p-isothiocyanatobenzyl)-5, 6-cyclohexano-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetate (CHX-DOTA) are reported. These chelating agents were compared to 2-(p-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (C-DOTA) and 1, 4, 7, 10-Tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N, N, N-tris(acetic acid) cyclododecane (PA-DOTA) as their 177 Lu radiolabeled conjugates with Herceptin™. In vitro stability of the immunoconjugates radiolabeled with 177 Lu was assessed by serum stability studies. The in vivo stability of the radiolabeled immunoconjugates and their targeting characteristics were determined by biodistribution studies in LS-174T xenograft tumor-bearing mice. Relative radiolabeling rates and efficiencies were determined for all four immunoconjugates. Insertion of the 1B4M moiety into the DOTA backbone increases radiometal chelation rate and provides complex stability comparable to C-DOTA and PA-DOTA while the CHX-DOTA appears to not form as stable a 177 Lu complex while exhibiting a substantial increase in formation rate. The 1B4M-DOTAmay have potential for radioimmunotherapy applications. Published by Elsevier Inc. All rights reserved. Keywords: DOTA; Bifunctional Chelating Agent; Lutetium-177; monoclonal antibody; biodistribution; stability 1. Introduction Use of radiolabeled monoclonal antibodies continues to be an attractive and burgeoning area of investigation as evidenced by the recent approval of the anti-CD20 targeting Zevalin ( 90 Y radiolabeled-Rituxan) for the treatment of non-Hodgkins lymphoma (NHL) [15]. One critical variable that influences the effectiveness of radioimmunotherapy (RIT) is the choice of the radionuclide and its associated emission characteristics [30]. Equally important is the choice of the chemical means by which the radionuclide is bound to the protein [11]. Therapy of solid tumors has been exacted with  - -emitting radionuclides such as 90 Y (T 1/2  2.67 d, E total  939.1 keV) and 177 Lu (T 1/2  6.71 d, E total  146.7 keV) [33]. The optimal range of the  - particles emitted by these isotopes for effective killing of cells, as defined by the physical properties of range and energy deposition, has been reported to be 28 – 42 mm and 1.2–3.0 mm, respectively, and the majority of energy deposition does not occur immediately along the emission track [24]. For RIT applications, 90 Y or 177 Lu must be linked as a metal complex to a monoclonal antibody (mAb) or immu- noprotein via a suitable bifunctional chelating agent that possesses acceptable thermodynamic and kinetic stability to minimize release of the isotope and hence in vivo toxicity [11]. Derivatives of 1, 4, 7, 10-tetraazacyclododecane-N, N,N,N-tetraacetic acid (DOTA) conjugated to monoclo- nal antibodies have been shown to form exceedingly stable complexes with the lanthanide metal ion radionuclides for in vivo applications [20,23,27]. This has resulted in the develop- ment of several bifunctional derivatives of DOTA for radio- labeling proteins, including 2-(p-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (C-DOTA) [20,23,27] and 1, 4, 7, 10-tetraaza-N-(1-car- boxy-3-(4-nitrophenyl)propyl)-N,N,N-tris(acetic acid) * Corresponding author. E-mail address: martinwb@mail.nih.gov (M.W. Brechbiel). Nuclear Medicine and Biology 30 (2003) 581–595 www.elsevier.com/locate/nucmedbio 0969-8051/03/$ – see front matter Published by Elsevier Inc. All rights reserved. doi:10.1016/S0969-8051(03)00033-7